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1.
An acidic or basic amino acid at position 26 of the b subunit of Escherichia coli F1F0-ATPase impairs membrane proton permeability: suppression of the uncF469 nonsense mutation. 总被引:2,自引:1,他引:1
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The uncF469 allele differed from normal in that a G----A base change occurred at nucleotide 77 of the uncF gene, resulting in a TAG stop codon rather than the tryptophan codon TGG. Two partial revertant strains were isolated which retained the uncF469 allele but formed a partially functional b-subunit, due to suppression of the uncF469 nonsense mutation. From the altered isoelectric points of the b-subunits from these strains, it was concluded that the suppressor gene of partial revertant strain AN1956 inserts an acidic amino acid for the TAG codon, and that the suppressor gene of partial revertant strain AN1958 inserts a basic amino acid. The membranes of both partial revertant strains showed impaired permeability to protons on removal of F1-ATPase. The membranes of both strains, however, were able to carry out oxidative phosphorylation, and the ATPase activities of both were resistant to the inhibitor dicyclohexylcarbodiimide. 相似文献
2.
Complementation between uncF alleles affecting assembly of the F1F0-ATPase complex of Escherichia coli. 总被引:1,自引:0,他引:1
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A mutant affected in the b subunit (coded by the uncF gene) of the F1F0-ATPase in Escherichia coli was isolated by a localized mutagenesis procedure in which a plasmid carrying the unc genes was mutagenized in vivo. The biochemical properties of cells carrying the uncF515 allele were examined in a strain carrying the allele on a multicopy plasmid and a mutator-induced polar unc mutation on the chromosome. The strain carrying the mutant unc allele was uncoupled with respect to oxidative phosphorylation. Membrane-bound ATPase activity was very low or absent, and membranes were somewhat proton permeable. It was concluded that the F0 sector was assembled. Determination of the DNA sequence of the uncF515 allele showed it differed from wild type in that a G----A substitution occurred at position 392, resulting in glycine being replaced by aspartate at position 131. Genetic complementation tests indicated that the uncF515 allele complemented the uncF476 allele (Gly 9----Asp). Two-dimensional gel electrophoresis of membrane preparations indicated that the uncF515 and uncF476 alleles interrupted assembly of the F1F0-ATPase at different stages. 相似文献
3.
4.
Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits. 总被引:16,自引:10,他引:6
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G B Cox J A Downie L Langman A E Senior G Ash D R Fayle F Gibson 《Journal of bacteriology》1981,148(1):30-42
A strain of Escherichia coli (AN1007) carrying the polar uncD436 allele which affects the operon coding for the F1-F0 adenosine triphosphatase (ATPase) complex was isolated and characterized. The uncD436 allele affected the two genes most distal to the operon promoter, i.e., uncD and uncC. Although the genes coding for the F0 portion of the ATPase complex were not affected in strains carrying this mutant allele, the lack of reconstitution of washed membranes by normal F1 ATPase suggested that a functional F0 might not be formed. This conclusion was supported by the observation that the 18,000-molecular-weight F0 subunit, coded for by the uncF gene, was absent from the membranes. Plasmid pAN36 (uncD+C+), when inserted into a strain carrying the uncD436 allele, resulted in the incorporation of the 18,000-molecular-weight F0 subunit into the membrane. A further series of experiments with Mu-induced polarity mutants, with and without plasmid pAN36, showed that the formation of both the alpha- and beta-subunits of F1 ATPase was an essential prerequisite to the incorporation into the membrane of the 18,000-molecular-weight F0 subunit and to the formation of a functional F0. Examination of the polypeptide composition of membranes from various unc mutants allowed a sequence for the normal assembly of the F1-F0 ATPase complex to be proposed. 相似文献
5.
Targeted mutagenesis of the b subunit of F1F0 ATP synthase in Escherichia coli: Glu-77 through Gln-85.
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Subunit b of Escherichia coli F1F0 ATP synthase contains a large hydrophilic region thought to be involved in the interaction between F1 and F0. Oligonucleotide-directed mutagenesis was used to evaluate the functional importance of a segment of this region from Glu-77 through Gln-85. The mutagenesis procedure employed a phagemid DNA template and a doped oligonucleotide primer designed to generate a predetermined collection of missense mutations in the target segment. Sixty-one mutant phagemids were identified and shown to contain nucleotide substitutions encoding 37 novel missense mutations. Mutations were isolated singly or in combinations of up to four mutations per recombinant phagemid. F1F0 ATP synthase function was studied by mutant phagemid complementation of a novel E. coli strain in which the uncF (b) gene was deleted. Complementation was assessed by observing growth on solid succinate minimal medium. Many phagemid-encoded uncF (b) gene mutations in the targeted segment resulted in growth phenotypes indistinguishable from those of strains expressing the native b subunit, suggesting abundant F1F0 ATP synthase activity. In contrast, several specific mutations were associated with a loss of enzyme function. Phagemids specifying the Ala-79----Pro, Arg-82----Pro, Arg-83----Pro, or Gln-85----Pro mutation failed to complement uncF (b) gene-deficient E. coli. F1F0 ATP synthase displayed the greatest sensitivity to mutations altering a single site in the target segment, Ala-79. The evidence suggests that Ala-79 occupies a restricted position in the enzyme complex. 相似文献
6.
A mutation affecting a second component of the F0 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12. The uncC424 allele. 总被引:23,自引:0,他引:23
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A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. The new mutant strain has a similar phenotype to the uncB mutant described previously; results from reconstitution experiments in vitro indicate that the new mutation also affects a component of the F0 portion of the Mg2+-stimulated adenosine triphosphatase. A method was developed to incorporate mutant unc alleles into plasmids. Partial diploid strains were prepared in which the uncB402 allele was incorporated into the plasmid and the new unc mutation into the chromosome, or vice versa. Complementation between the mutant unc alleles was indicated by growth on succinate, growth yields on glucose, ATP-dependent transhydrogenase activities, ATP-induced atebrin-fluorescence quenching and oxidative-phosphorylation measurements. The gene in which the new mutation occurs is therefore distinct from the uncB gene, and the mutant allele was designated uncC424. 相似文献
7.
Lonjers ZT Dickson EL Chu TP Kreutz JE Neacsu FA Anders KR Shepherd JN 《Journal of bacteriology》2012,194(5):965-971
Rhodoquinone (RQ) is a required cofactor for anaerobic respiration in Rhodospirillum rubrum, and it is also found in several helminth parasites that utilize a fumarate reductase pathway. RQ is an aminoquinone that is structurally similar to ubiquinone (Q), a polyprenylated benzoquinone used in the aerobic respiratory chain. RQ is not found in humans or other mammals, and therefore, the inhibition of its biosynthesis may provide a novel antiparasitic drug target. To identify a gene specifically required for RQ biosynthesis, we determined the complete genome sequence of a mutant strain of R. rubrum (F11), which cannot grow anaerobically and does not synthesize RQ, and compared it with that of a spontaneous revertant (RF111). RF111 can grow anaerobically and has recovered the ability to synthesize RQ. The two strains differ by a single base pair, which causes a nonsense mutation in the putative methyltransferase gene rquA. To test whether this mutation is important for the F11 phenotype, the wild-type rquA gene was cloned into the pRK404E1 vector and conjugated into F11. Complementation of the anaerobic growth defect in F11 was observed, and liquid chromatography-time of flight mass spectrometry (LC-TOF-MS) analysis of lipid extracts confirmed that plasmid-complemented F11 was able to synthesize RQ. To further validate the requirement of rquA for RQ biosynthesis, we generated a deletion mutant from wild-type R. rubrum by the targeted replacement of rquA with a gentamicin resistance cassette. The ΔrquA mutant exhibited the same phenotype as that of F11. These results are significant because rquA is the first gene to be discovered that is required for RQ biosynthesis. 相似文献
8.
A mutation in the Neisseria gonorrhoeae rfaD homolog results in altered lipooligosaccharide expression. 总被引:4,自引:1,他引:3
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The gonococcal lsi-6 locus was cloned and shown by DNA sequence analysis to have homology with the E. coli rfaD gene, which encodes ADP-L-glycero-D-mannoheptose epimerase. This enzyme is involved in the biosynthesis of the lipopolysaccharide precursor ADP-L-glycero-D-mannoheptose. A site-directed frameshift mutation in lsi-6 was constructed by PCR amplification and introduced into the chromosome of Neisseria gonorrhoeae MS11 P+ by transformation. The lipooligosaccharides (LOS) of mutant and parental strains were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lsi-6 mutant produced LOS components with apparent molecular masses of 2.6 and 3.6 kDa as compared with a 3.6-kDa band of the MS11 P+ strain. The parental LOS phenotype was expressed when a revertant was constructed by transformation of the cloned wild-type gene into the lsi-6 mutant. The immunoreactivity of LOS from parental and constructed strains was examined by SDS-PAGE and Western blotting. Only the parental and reconstructed wild-type strains produced a 3.6-kDa LOS component that reacted with monoclonal antibody 2-1-L8. These results suggest that the lsi-6 locus is involved in gonococcal LOS biosynthesis and that the nonreactive mutant 3.6-kDa LOS component contains a conformational change or altered saccharide composition that interferes with immunoreactivity. 相似文献
9.
Identification of Escherichia coli ubiB, a gene required for the first monooxygenase step in ubiquinone biosynthesis 总被引:2,自引:0,他引:2
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Poon WW Davis DE Ha HT Jonassen T Rather PN Clarke CF 《Journal of bacteriology》2000,182(18):5139-5146
It was recently discovered that the aarF gene in Providencia stuartii is required for coenzyme Q (CoQ) biosynthesis. Here we report that yigR, the Escherichia coli homologue of aarF, is ubiB, a gene required for the first monooxygenase step in CoQ biosynthesis. Both the P. stuartii aarF and E. coli ubiB (yigR) disruption mutant strains lack CoQ and accumulate octaprenylphenol. Octaprenylphenol is the CoQ biosynthetic intermediate found to accumulate in the E. coli strain AN59, which contains the ubiB409 mutant allele. Analysis of the mutation in the E. coli strain AN59 reveals no mutations within the ubiB gene, but instead shows the presence of an IS1 element at position +516 of the ubiE gene. The ubiE gene encodes a C-methyltransferase required for the synthesis of both CoQ and menaquinone, and it is the 5' gene in an operon containing ubiE, yigP, and ubiB. The data indicate that octaprenylphenol accumulates in AN59 as a result of a polar effect of the ubiE::IS1 mutation on the downstream ubiB gene. AN59 is complemented by a DNA segment containing the contiguous ubiE, yigP, and ubiB genes. Although transformation of AN59 with a DNA segment containing the ubiB coding region fails to restore CoQ biosynthesis, transformation with the ubiE coding region results in a low-frequency but significant rescue attributed to homologous recombination. In addition, the fre gene, previously considered to correspond to ubiB, was found not to be involved in CoQ biosynthesis. The ubiB gene is a member of a predicted protein kinase family of which the Saccharomyces cerevisiae ABC1 gene is the prototypic member. The possible protein kinase function of UbiB and Abc1 and the role these polypeptides may play in CoQ biosynthesis are discussed. 相似文献
10.
In the sexual reproduction of Paramecium tetraurelia, the somatic nucleus (macronucleus) undergoes massive genomic rearrangement, including gene amplification and excision of internal eliminated sequences (IESs), in its normal developmental process. Strain d4-662, one of the pawn mutants, is a behavioral mutant of P. tetraurelia that carries a recessive allele of pwB662. ThepwB gene in the macronucleus of the strain has an insertion of the IES because a base substitution within the IES prevents its excision during gene rearrangement. Cultures of this strain frequently contain cells reverting to the wild type in the behavioral phenotype. The mutant and revertant cells maintained stable clonal phenotypes under the various environmental conditions examined unless they underwent sexual reproduction. After sexual reproduction, both mutant and revertant produced 2.7-7.1% reverted progeny. A molecular analysis performed on the macronuclear DNA of the mutant and revertant of d4-662 showed that much less than 1% of the mutant IES was precisely excised at every sexual reproduction of the strain. Therefore, the alternative phenotype of strain d4-662 seems to be caused by an alternative excision of the mutant IES. 相似文献
11.
A novel rho promoter::Tn10 mutation suppresses and ftsQ1(Ts) missense mutation in an essential Escherichia coli cell division gene by a mechanism not involving polarity suppression. 总被引:2,自引:1,他引:1
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An extragenic suppressor of the Escherichia coli cell division gene ftsQ1(Ts) was isolated. The suppressor is a Tn10 insertion into the -35 promoter consensus sequence of the rho gene, designated rho promoter::Tn10. The ftsQ1(Ts) mutation was also suppressed by the rho-4 mutant allele. The rho promoter::Tn10 strain does not exhibit rho mutant polarity suppressor phenotypes. In addition, overexpression of the ftsQ1(Ts) mutation does not reverse temperature sensitivity. Furthermore, DNA sequence analysis of the ftsQ1(Ts) allele revealed that the salt-remediable, temperature-sensitive phenotype arose from a single missense mutation. The most striking phenotype of the rho promoter::Tn10 mutant strain is an increase in the level of negative supercoiling. On the basis of these observations, we conclude that the ftsQ1(Ts) mutation may be suppressed by a change in supercoiling. 相似文献
12.
Studies of N-ethyl-N-nitrosourea (ENU)-induced mutagenesis with a tyrosine auxotroph of Escherichia coli revealed a new type of revertant. This mutant strain was interesting because: (i) it was not a true revertant of the nonsense (ochre) defect nor a tRNA suppressor mutation; and (ii) it was induced by ENU to greater extent in a UmuC-defective host. Genetic mapping located the probable mutation to a region of the E. coli chromosome containing a newly described gene called tas. To investigate this mutation, the upstream region of the tas gene from both wild-type and mutant cells was cloned into a promoterless lacZ expression vector and recombined onto a lambda bacteriophage. Recombinant bacteriophage were inserted into the bacterial chromosome and beta-galactosidase (betaGal) assays were performed. These assays revealed an almost three-fold greater expression of betaGal from the mutant DNA than from the wild-type DNA. Sequence analysis of the region directly upstream of the tas gene revealed a G:C to A:T transition at base number 2263 (numbering based on GenBank Accession #AE000367), located within a potential promoter site. Further sequencing indicated no other mutations within the 1454bp region analyzed; however, there were several nucleotide differences seen in our B/r strain of E. coli, when compared with the published E. coli K-12 sequence. A total of 10 base differences were discovered; one in mutH, six within a potential open reading frame (ORF-o237) and three in non-coding regions. Yet, none of the changes altered the predicted amino acid sequences. These results provide evidence of a mechanism for increased expression of the novel gene tas and support the neutral drift hypothesis for the evolution of DNA sequences. 相似文献
13.
The mutant allele (uncA401) of the gene for the alpha subunit of Escherichia coli F1-ATPase was cloned from the total DNA of the mutant AN120 on a hybrid plasmid pAN120. Determination of the DNA sequence of the alpha subunit gene from pAN120 revealed a single base change of cytosine at nucleotide residue 1118 to thymine and indicated that serine 373 was replaced by phenylalanine. It has been reported that the mutant F1 is defective in a step of steady-state catalysis, whereas its single turnover process is normal (Kanazawa, H., Noumi, T., Matsuoka, I., Hirata T., and Futai, M. (1984) Arch. Biochem. Biophys. 228, 258-269). Thus, we concluded that serine 373 in the alpha subunit is essential for steady-state catalysis by F1-ATPase. 相似文献
14.
CooB is required for assembly but not transport of CS1 pilin 总被引:6,自引:0,他引:6
J. R. Scott J. C. Wakefield P. W. Russell P. E. Orndorff B. J. Froehlich 《Molecular microbiology》1992,6(3):293-300
15.
Laura J. F. Hefta Alfred S. Lewin Bertrand Daignan-Fornier Monique Bolotin-Fukuhara 《Molecular & general genetics : MGG》1987,207(1):106-113
Summary Yeast strain 990 carries a mutation mapping to the oli1 locus of the mitochondrial genome, the gene encoding ATPase subunit 9. DNA sequence analysis indicated a substitution of valine for alanine at residue 22 of the protein. The strain failed to grow on nonfermentable carbon sources such as glycerol at low temperature (20°C). At 28°C the strain grew on nonfermentable carbon sources and was resistant to the antibiotic oligomycin. ATPase activity in mitochondria isolated from 990 was reduced relative to the wild-type strain from which it was derived, but the residual activity was oligomycin resistant. Subunit 9 (the DCCD-binding proteolipid) from the mutant strain exhibited reduced mobility in SDS-polyacrylamide gels relative to the wild-type proteolipid. Ten revertant strains of 990 were analyzed. All restored the ability to grow on glycerol at 20°C. Mitotic segregation data showed that eight of the ten revertants were attributable to mitochondrial genetic events and two were caused by nuclear events since they appeared to be recessive nuclear suppressors. These nuclear mutations retained partial resistance to oligomycin and did not alter the electrophoretic behavior of subunit 9 or any other ATPase subunit. When mitochondrial DNA from each of the revertant strains was hybridized with an oligonucleotide probe covering the oli1 mutation, seven of the mitochondrial revertants were found to be true revertants and one a second mutation at the site of the original 990 mutation. The oli1 gene from this strain contained a substitution of glycine for valine at residue 22. The proteolipid isolated from this strain had increased electrophoretic mobility relative to the wild-type proteolipid.Abbreviations DCCD
dicyclohexylcarbodiimide
- SDS
sodium dodecyl sulfate
- PMSF
phenylmethylsulfonyl fluoride
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonate
- SMP
submitochondrial particles
- mit-
mitochondrial point mutant 相似文献
16.
Escherichia coli strain AN718 contains the alpha S373F mutation in F1F0-ATP synthase which blocks ATP synthesis (oxidative phosphorylation) and steady-state F1-ATPase activity. The revertant strain AN718SS2 containing the mutation alpha C373 was isolated and shown to confer a phenotype of higher growth yield than that of the wild type in liquid medium containing limiting glucose, succinate, or LB. Purified F1 from strain AN718SS2 was found to have 30% of wild-type steady-state ATPase activity and 60% of wild-type oxidative phosphorylation activity. Azide sensitivity of ATPase activity and ADP-induced enhancement of bound aurovertin fluorescence, both of which are lost in alpha S373F mutant F1, were regained in alpha C373 F1. N-Ethylmaleimide (NEM) inactivated alpha C373 F1 steady-state ATPase potently but had no effect on unisite ATPase. Complete inactivation of alpha C373 F1 steady-state ATPase corresponded to incorporation of one NEM per F1 (mol/mol), in just one of the three alpha subunits. NEM-inactivated enzyme showed azide-insensitive residual ATPase activity and loss of ADP-induced enhancement of bound aurovertin fluorescence. The data confirm the view that placement at residue alpha 373 of a bulky amino acid side-chain (phenylalanyl or NEM-derivatized cysteinyl) blocks positive catalytic cooperativity in F1. The fact that NEM inhibits steady-state ATPase when only one alpha subunit of three is reacted suggests a cyclical catalytic mechanism. 相似文献
17.
SUC1 gene of Saccharomyces: a structural gene for the large (glycoprotein) and small (carbohydrate-free) forms of invertase. 总被引:4,自引:4,他引:0
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Saccharomyces cerevisiae revertant strain D10-ER1 has been shown to contain thermosensitive forms of the large (glycoprotein) and small (carbohydrate-free) invertases and a very low level of the small enzyme, along with a wild-type level of the large form (T. Mizunaga et al., Mol. Cell. Biol. 1:460-468, 1981). These characteristics cosegregated in crosses of the revertant strain with wild-type sucrose-fermenting (SUC1) or nonfermenting (suc0) strains. In addition, there is tight linkage between sucrose and maltose fermentation in revertant D10-ER1 (characteristic of the SUC1 and MAL1 genes). From this we infer that a single reversion event is responsible for the several changes observed in D10-ER1, and that this mutation maps within or very close to the SUC1 gene present in the ancestor strain 4059-358D. The revertant SUC1 allele in D10-ER1 (termed SUC1-R1) was expressed independently of the wild-type SUC1 gene when both were present in diploid cells. Diploids carrying only the wild-type or the mutant genes synthesized invertases with the characteristics of the parental Suc+ haploids. The possibility that a modifier gene was responsible for the alterations in the invertases of revertant D10-ER1 was ruled out by appropriate crosses. We conclude that SUC1 is a structural gene that codes for both the large and the small forms of invertase and suggest that SUC2 through SUC5 are structural genes as well. 相似文献
18.
19.
Genetic mapping of mutations using phenotypic pools and mapped RAPD markers. 总被引:4,自引:0,他引:4
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Genetic markers facilitate the study of inheritance and the cloning of genes by genetic approaches. Molecular markers detect differences in DNA sequence, and are thus less ambiguous than phenotypic markers, which require gene expression. We have demonstrated a molecular approach to the mapping of mutant genes using RAPD markers and pooling of individuals based on phenotype. To map genes by phenotypic pooling a strain carrying a mutation is crossed to a strain that is homozygous for the wild-type allele of the corresponding gene. A set of primers corresponding to mapped RAPDs distributed throughout the genome and in coupling phase with respect to the wild type parent is then used to amplify DNA from wild type and mutant pools of F2 individuals. Linkage between the mutant gene and the RAPD markers is visualized by the absence of the corresponding RAPD DNA bands in the mutant pool. We developed a mathematical model for calculating the probability of linkage between RAPDs and target genes and we successfully tested this approach with the model plant Arabidopsis thaliana. 相似文献
20.
In an earlier study, the ATP10 gene of Saccharomyces cerevisiae was shown to code for an inner membrane protein required for assembly of the F(0) sector of the mitochondrial ATPase complex (Ackerman, S., and Tzagoloff, A. (1990) J. Biol. Chem. 265, 9952-9959). To gain additional insights into the function of Atp10p, we have analyzed a revertant of an atp10 null mutant that displays partial recovery of oligomycin-sensitive ATPase and of respiratory competence. The suppressor mutation in the revertant has been mapped to the OLI2 locus in mitochondrial DNA and shown to be a single base change in the C-terminal coding region of the gene. The mutation results in the substitution of a valine for an alanine at residue 249 of subunit 6 of the ATPase. The ability of the subunit 6 mutation to compensate for the absence of Atp10p implies a functional interaction between the two proteins. Such an interaction is consistent with evidence indicating that the C-terminal region with the site of the mutation and the extramembrane domain of Atp10p are both on the matrix side of the inner membrane. Subunit 6 has been purified from the parental wild type strain, from the atp10 null mutant, and from the revertant. The N-terminal sequences of the three proteins indicated that they all start at Ser(11), the normal processing site of the subunit 6 precursor. Mass spectral analysis of the wild type and mutants subunit 6 failed to reveal any substantive difference of the wild type and mutant proteins when the mass of the latter was corrected for Ala --> Val mutation. These data argue against a role of Atp10p in post-translational modification of subunit 6. Although post-translational modification of another ATPase subunit interacting with subunit 6 cannot be excluded, a more likely function for Atp10p is that it acts as a subunit 6 chaperone during F(0) assembly. 相似文献